1. Field of the Invention
The present invention relates to the isolation and propagation of a novel human herpesvirus derived from AIDS-associated Kaposi's Sarcoma cells, diagnostic methods using the same, methods for screening for anti-viral drugs using the same and vaccines against Kaposi's Sarcoma.
2. Discussion of the Background
Kaposi's sarcoma (KS) was originally described in the late 1800's as a rare and relatively benign neoplasm of elderly men of Jewish or Mediterranean descent. Today, KS is recognized as the most common malignancy in AIDS patients, affecting approximately 20% of human immunodeficiency virus-1 (HIV-1)-positive patients (1-3). AIDS-related KS (also known as epidemic KS) is clinically more aggressive than other forms of KS, including classical (Mediterranean), African (endemic), and iatrogenic immunosuppressive-drug associated with organ transplantation [reviewed in (4,5)]. While often presenting in the skin of HIV-1 positive patients, AIDS-KS lesions can involve internal organs, particularly the lungs and gastrointestinal tract, resulting in severe and potentially fatal hemorrhagic disease (6).
KS is characterized histologically by a proliferation of spindle-shaped tumor cells mixed with endothelial cells forming blood vessels (angiogenesis), fibroblasts, dermal dendrocytes, and an inflammatory cell infiltrate (7). The origin of the spindle-shaped KS tumor cells has been difficult to define with certainty, but is thought to derive from either endothelial or vascular smooth muscle cells (8-11). In addition, a variety of immune or inflammatory cytokines and oncogenes or viral proteins have been found in association with these lesions (11-15). While the etiologic agent causing KS is unclear, epidemiological data has suggested that an infectious agent could potentially spread the disease through sexual contact (16). Although several viruses, including cytomegalovirus, hepatitis B virus, and human papillomavirus were found in KS patients, no single agent was found consistently in all patient lesions until recently when Chang, Moore, and colleagues demonstrated that over 90% of AIDS-KS tissue samples were positive for herpesvirus-like DNA sequences (17). These DNA sequences were homologous to, but distinct from, minor capsid and tegument proteins of Epstein-Barr virus (EBV) and herpesvirus saimiri (17), and defined a putative new member of the gammaherpesvirus family which is currently referred to as KS-associated herpesvirus, KSHV, or human herpesvirus 8, HHV-8. Since then, several different laboratories have demonstrated the presence of this viral sequence in tissue from KS patients with classic, African endemic and AIDS-associated KS (18-20) as well as patients with body-cavity-based lymphomas (21,22).
It has remained uncertain, however, whether this new herpesvirus was replication-competent or represented a replication-defective, adventitious virus present in KS tissue. To date, although the presence of this virus has been noted in B lymphoma cells which also contain EBV, it has not been possible to demonstrate viral replication in vitro.
Since the publication of a DNA sequence associated with Kaposi sarcoma, several reports have either supported (18-20,23-25) or refuted (26,27) the notion that this novel DNA virus was important in KS. To date, it had not been possible to propagate this putative virus or even to maintain it in cell culture in the absence of other known herpesviruses, making it difficult to characterize its transmissibility and its host cell range, and to develop anti-viral and diagnostic reagents.
It is therefore desirable to isolate intact human herpesvirus from Kaposi's sarcoma cells and to propagate the same in vitro. The earliest studies in this field defined a novel viral DNA sequence associated with Kaposi's sarcoma lesions (Chang et al., Science (1994) 266:1865-1869). Although the sequence suggested that it was a member of the gamma herpesvirus family, it has not been possible to address the question of whether this represents a true replicating virus or a helper virus associated with another pathogen that might cause the disease. More recently, the presence of the virus DNA sequence was noted in a B cell lymphoma in a persistent form (22). Unfortunately, this line contained a second herpesvirus, EBV, which did not allow definitive isolation or identification of the KS-associated virus. In addition, it did not provide a means to replicate or propagate the virus which was carried in these cells but could not be amplified. The ability now to propagate and clone this virus allows specific diagnostic tests to be developed, by allowing the definition of the full range of the DNAs, RNAs and proteins in the native virus, by permitting the generation of antibodies to diagnose the virus, and by providing a means to establish its epidemiologic association with the disease. It also establishes a method to generate a prototype virus which may be useful for the development of a vaccine to prevent this disease. This isolation of this virus will also make it possible to fulfill Koch's postulates in animal models, showing that it can transmit the genetic information which causes the disease. Finally, it provides a critical assay system for the replication of the virus and allows for large scale screening of antiviral compounds that inhibit viral replication in vitro. Without the ability to replicate the virus, these goals could not be achieved. The present invention provides a method to establish and propagate replication-competent virus which will make these goals possible and likely to succeed. It thus represents a major advance in the field which was not obvious and was widely recognized as needed.